Biocompatible material composition adaptable to diverse therapeutic indications

ABSTRACT

A biocompatible material genus serves as the foundation for multiple material composition species, each adapted to a specific therapeutic indication.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/520,856, filed Mar. 7, 2000, now U.S. Pat. No. 6,899,889 which is acontinuation-in-part of U.S. patent application Ser. No. 09/283,535,filed Apr. 1, 1999, now U.S. Pat. No. 6,458,147, which is itself acontinuation-in-part of U.S. patent application Ser. No. 09/188,083,filed Nov. 6, 1998, now U.S. Pat. No. 6,371,975.

FIELD OF THE INVENTION

The invention generally relates to the composition of biocompatiblematerials and their application to body tissue to affect desiredtherapetic results.

BACKGROUND OF THE INVENTION

There are many therapeutic indications today that pose problems in termsof technique, cost efficiency, or efficacy, or combinations thereof.

For example, following an interventional procedure, such as angioplastyor stent placement, a 5 Fr to 8 Fr arteriotomy remains. Typically, thebleeding from the arteriotomy is controlled through pressure applied byhand, by sandbag, or by C-clamp for at least 30 minutes. While pressurewill ultimately achieve hemostasis, the excessive use and cost of healthcare personnel is incongruent with managed care goals.

As another example, blood leaks from a variety of different tissues arecommon during surgical procedures. Examples include following trauma toor resection of the liver, spleen, or kidney, vascular anastomoses, andbone bleeding during sternotomy. Presently, the surgeon has a limitednumber of options to control bleeding, typically pressure, thrombin,fibrin glue, bone wax, and/or collagen sponge.

Likewise, controlling air leaks from lung tissue is difficult to achieveduring thoracic procedures. Examples include lung resections and lungvolume reduction surgery. Presently, the surgeon has a limited number ofoptions to control air leaks, typically a chest tube is required toremove air from the thoracic cavity. The presence of a chest tubeextends the stay of the patient in the hospital. If the air leaks couldbe sealed at the time of surgery, the patient would be able to bedischarged sooner.

Similarly, controlling liquid leaks from tissue is difficult to achieveduring surgical procedures. Examples include dural leaks and lymph fluidleaks during surgical procedures. Typically, the surgeon does notcontrol dural leaks due to the lack of an effective dural substitute,potentially increasing the risk of transmission of infectious agents.Controlling solid leaks from tissue is likewise difficult to achieveduring surgical procedures. Examples include bowel leaks during surgicalprocedures. Typically, the surgeon controls bowel leaks by addingadditional sutures until the leak is no longer observed.

As another example, adhesions are abnormal, fibrous connections oftissues that are not normally connected. Adhesions are formed as a partof the normal wound healing response of tissue, however they can resultin infertility and pain. Several products are available for use by thesurgeon to prevent the formation of adhesions, however the efficacy ofthe marketed products has not been conclusively demonstrated.

Likewise, tissue voids can be created by a variety of procedures. Forexample, the ABBITM system, marketed by United States Surgical Company,is a minimally invasive breast biopsy system that cores out breasttissue for analysis by a pathologist. The cores range in size from fiveto twenty millimeters in diameter. Following the removal of the core, atissue void is created and the surrounding tissue oozes blood into thevoid.

Various tissues can also be augmented to create a more desiredappearance. For example, an injectable bovine collagen, marketed byInamed Corporation, can be used to reduce the appearance of facialwrinkles or create the appearance of fuller lips. While the treatment iseffective, the persistence is brief.

The treatment of arterio-venous malformations (AVM's) and aneurysmsprovide further examples. AVM's are tangled masses of blood vessels thatare neither arteries nor veins, commonly found in the brain, possiblyleading to hemorragic stroke. Clinically, AVM's are treated by surgicalremoval. Before removal, the AVM must be embolized to preventuncontrolled bleeding. Aneurysms are abnormal widening of portions ofblood vessels, leading to an increased chance of rupture. Clinically,aneurysms are treated by surgical removal, stent-grafting, or coils.Another possible treatment modality is to fill the ballooned section ofthe blood vessel with a biomaterial, protecting and strengthening thediseased tissue.

There is also an increasing trend towards site-specific delivery ofpharmaceuticals and vectors. The main advantage is high dose delivery atthe diseased tissue, but a low systemic dose. For example, a depotfilled with anti-cancer agents can be placed directly on a tumor. Theareas surrounding the depot have a high concentration of the anti-canceragent, but the systemic dose is low, minimizing side effects.

Cells, as well as pharmaceuticals and vectors, can be likewise deliveredto a diseased tissue site. The cells could be genetically modified,autologous, or derived from other sources.

There remains a demand for biomaterials that improve the technique, costefficiency, and efficacy of these and other therapeutic indications.

SUMMARY OF THE INVENTION

One aspect of the invention provides a biocompatible genus materialcomposition upon which a diverse family of biocompatible materialcomposition species can be created. The species are remarkably welladapted to specific therapeutic indications, although the therapeuticindications themselves differ significantly.

In one embodiment, the genus biocompatible material comprises a mixtureof a protein solution and a polymer solution. The polymer solutionincludes a derivative of a hydrophilic polymer with a functionality ofat least three. Upon mixing, the protein solution and the polymersolution cross-link to form a non-liquid, three-dimensional network.

In one embodiment, the network degrades over time back to a liquid form.In this embodiment, the polymer includes a degradation control regionselected to achieve a desired degradation period. The degradationcontrol region can selected to form different species, each having adifferent degradation period. The degradation periods can vary, e.g.,within a range of between about 1 day to greater than 500 days.

In one embodiment, the polymer includes a cross-linking group selectedto achieve a desired cross-linking period. The cross-linking group canbe selected to achieve different species, each having its own desiredcross-linking period. The cross-linking periods can vary, e.g., within arange of from less than one second to greater than 10 hours.

In one embodiment, the polymer includes both a degradation controlregion and a cross-linking group. In this embodiment, each region can beindividually selected to form different species, each species having itsown period of degradation and cross-linking customized to achieve one ormore therapeutic objectives.

Another aspect of the invention provides systems making use of thediverse species for different therapeutic indications. Each systemincludes instructions for forming a mixture of the protein solution andpolymer solution and for applying the mixture to accomplish a particulartherapeutic objective. The particular therapeutic objective can be,e.g., sealing a vascular puncture site, sealing tissue from blood or gasor liquid leaks, sealing tissue from solid leaks, preventing postoperative adhesions, repairing a tissue void, augmenting tissue,embolizing an arterio-venous malformation, filling an aneurysm,delivering a pharmaceutical, or delivering cells.

Another aspect of the invention provides a biocompatible materialcomprising a mixture of a protein solution and a polymer solution which,upon mixing, cross-link to form a non-liquid, three-dimensional network.The material includes an agent that undergoes color change in responseto cross-linking of the mixture.

In one embodiment, the agent undergoes color change in response tochange in pH during cross-linking.

In one embodiment, the agent exhibits a first color when the mixture isin a liquid state and a second color, different than the first color,when the mixture forms the non-liquid, three-dimensional network.

In one embodiment, the agent exhibits a first color when the mixture isin transition between a liquid state and the non-liquid, threedimensional network, and a second color, different than the first color,when the mixture forms the non-liquid, three-dimensional network.

Features and advantages of the inventions are set forth in the followingDescription and Drawings, as well as in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of a genus composition of a biocompatiblematerial that forms the basis for multiple composition species, eachwell adapted to a specific therapeutic indication;

FIG. 2 is a diagrammatic view of a system comprising first and secondfunctional kits useful for applying or delivering a given compositionspecies shown in FIG. 1 at the intended delivery site;

FIG. 3 is a plane view of the details of representative kits shown inFIG. 2, one kit containing the basic formative components of the genuscomposition, and the other kit containing a mixing/dispensing assemblyfor the species;

FIG. 4 is a perspective view of a representative mixing/dispensingassembly contained in the second kit shown in FIG. 3;

FIG. 5 is a flowchart illustrating a methodology for developing speciescompositions based upon the composition genus shown in FIG. 1; and

FIG. 6 is a graph showing the changes on pH as a given speciescomposition cross-links to form a solid three-dimensional network.

The invention may be embodied in several forms without departing fromits spirit or essential characteristics. The scope of the invention isdefined in the appended claims, rather than in the specific descriptionpreceding them. All embodiments that fall within the meaning and rangeof equivalency of the claims are therefore intended to be embraced bythe claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. Overview

FIG. 1 shows a genus composition 10 comprising a biocompatible material.The genus composition 10 is the basis for multiple material compositionspecies 12.

The material composition species 12 of the genus composition 10 shareseveral common fundamental characteristics, including:

(i) each composition species 12 is capable of being created in situ bymixing basic formative components, at least two of which are commonthroughout the genus;

(ii) the formative components, upon mixing, are capable of transformingfrom a liquid state to a biocompatible solid state (in a process called“gelation”);

(iii) after gelation, the solid composition exhibits desired mechanicalproperties including adhesive strength, cohesive strength,andelasticity;

(iv) after gelation, the solid composition is also capable oftransforming over time by physiological mechanisms from the solid stateto a biocompatible liquid state, which can be cleared by the body (in aprocess called “degradation”); and

(v) each of the characteristics (ii) to (iv) can be selectively andindependent controlled within wide physical ranges.

The common fundamental characteristics of the genus 10 give rise to adiverse family of biocompatible material composition species 12, havingdiffering rates of gelation, rates of degradation, and mechanicalproperties. The species are remarkably well adapted to specifictherapeutic indications, although the therapeutic indications themselvesdiffer significantly.

In the illustrated embodiment (see FIG. 1), the genus 10 is shown, byway of example, to contain twelve distinct species 12, each having acorresponding different therapeutic indication, as also listed in thefollowing Table.

TABLE 1 Biocompatible Material Composition Species and CorrespondingTherapeutic Indications Species Therapeutic Indication 1 SealingVascular Puncture Sites 2 Sealing Tissue From Blood Leaks 3 SealingTissue From Gas Leaks 4 Sealing Tissue From Liquid Leaks 5 SealingTissue From Solid Leaks 6 Preventing Post-Operative Adhesions 7 Repairof Tissue Voids 8 Augmentation of Tissue 9 Embolization ofArterio-Venous Malformations (AVM) 10 Filling of Aneurysms 11 Deliveryof Pharmaceuticals 12 Delivery of Cells

As FIG. 2 shows, the common fundamental characteristics of thecomposition genus 10 make possible the development of a family ofsystems 14 for applying or delivering the species 12 at the intendedtreatment site. Each delivery system 14 shares certain common features,because of characteristics common to the genus composition 10.Nevertheless, the delivery systems 14 also differ in specific respects,because each species 12 is tailored to meet the needs of a particulardesired therapeutic indication.

Due to the common fundamental characteristics (i) and (ii) of the genus10, each delivery system can be consolidated into two functional kits 16and 18. The first kit 16 contains the basic formative components 20 ofthe genus composition 10. The kit 16 stores the basic formativecomponents 20 in an unmixed condition prior to use. Certain aspects ofthe formative components 20 differ, according to the species 12 that thesystem 14 delivers or applies. Still, the fundamental characteristic ofholding the basic formative components 20 in an unmixed condition untilthe instance of use is common to all delivery systems 14 of the family.

The second kit 18 contains a mixing/dispensing assembly 22 for eachspecies 12. The mixing/dispensing assembly 22 brings the formativecomponents 20 into intimate mixing contact in liquid form. Themixing/dispensing assembly 22 dispenses the liquid mixture to theintended therapeutic site, where the liquid mixture transforms in situto a solid form. Among the delivery systems 14, certain aspects of themixing/dispensing assembly 22 differ, according to requirements of theparticular therapeutic indication. Still, the fundamental characteristicof the mixing of the formative components 20 for in-situ delivery iscommon to all delivery systems 14.

One or both kits 16 and 18 preferably includes instructions 24 forforming the liquid mixture and dispensing the liquid through themixing/dispensing assembly 22 to achieve the particular desiredtherapeutic indication.

II. The Genus Material Composition

In a preferred embodiment, the genus material composition 10 creates anon-liquid, three-dimensional network, termed a hydrogel. In thisembodiment, there are two fundamental formative components common to thegenus 10; namely, (i) a protein and (ii) a polymer.

The hydrogel is created by the reaction between one or more nucleophilic(electron donator) groups on one of the components (either the proteinor the polymer) and one or more electrophilic (electrode withdrawing)groups on the other one of the components. The polymer can include oneor more nucleophlic groups, such as amine groups (—NH2), or thiol groups(—SH), or one or more electrophilic groups, such as alcohol groups (—OH)or carboxyl groups (—COOH), or combinations thereof. Likewise, thesequence of amino acids forming the protein (which determines theprotein's bioactivity) can provide nucleophilic reactivity (e.g.,lysine, arginine, asparagine, glutamine, or cysteine), or can provideelectrophilic reactivity (e.g., aspartic acid, glutamic acid, serine,threonine, or tyrosine), or both. The amino acid sequence of the proteincan thus dictate the selection of the polymer, and vice versa.

The protein component most preferably takes the form of a proteinsolution with nucleophilic groups. The polymer component most preferablytakes the form of a solution of an electrophilic derivative of ahydrophilic polymer with a functionality of at least three. In thisarrangement, the electrophilic groups on the polymer react with thenucleophilic groups on the protein, to form the hydrogel.

Use of these two fundamental components permit the rates of gelation anddegradation to be accurately and predictably controlled, to create avariety of different species 12. Furthermore, a genus materialcomposition 10 based upon these two fundamental components possessesdesirable mechanical properties, which can also be accurately andselective manipulated among the species. The ability to accurately andselectively control the rate of gelation, the rate of degradation, andthe mechanical properties, allows the creation of diverse species, eachoptimized to meet the requirements of a particular therapeuticindication.

A. The Fundamental Protein Component

Suitable protein solutions for incorporation into the genus material 10include non-immunogenic, hydrophilic proteins. Examples include serum,serum fractions, and solutions of albumin, gelatin, antibodies,fibrinogen, and serum proteins. In addition, water soluble derivativesof hydrophobic proteins can be used. Examples include solutions ofcollagen, elastin, chitosan, and hyaluronic acid. In addition, hybridproteins with one or more substitutions, deletions, or additions in theprimary structure may be used.

Furthermore, the primary protein structure need not be restricted tothose found in nature. An amino acid sequence can be syntheticallydesigned to achieve a particular structure and/or function and thenincorporated into the genus material. The protein can be recombinantlyproduced or collected from naturally occurring sources.

The preferred protein solution is 25% human serum albumin, USP. Humanserum albumin is preferred due to its biocompatibility and its readyavailability.

B. The Fundamental Polymer Component

The fundamental polymer component of the genus material composition is ahydrophilic, biocompatible polymer that is electrophilically derivatizedwith a functionality of at least three. Examples include poly(ethyleneglycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidinone), poly(ethyloxazoline), and poly(ethyleneglycol)-co-poly(propylene glycol) block copolymers.

The fundamental polymer component is not restricted to syntheticpolymers, as polysaccharides, carbohydrates, and proteins could beelectrophilically derivatized with a functionality of at least three. Inaddition, hybrid proteins with one or more substitutions, deletions, oradditions in the primary structure may be used as the polymer component.In this arrangement, the protein's primary structure is not restrictedto those found in nature, as an amino acid sequence can be syntheticallydesigned to achieve a particular structure and/or function and thenincorporated into the material. The protein of the polymer component canbe recombinantly produced or collected from naturally occurring sources.

Preferably, the polymer component is comprised of poly(ethylene glycol)(PEG) with a molecular weight between 1,000 and 30,000 g/mole, morepreferably between 2,000 and 15,000, and most preferably between 10,000and 15,000 g/mole. PEG has been demonstrated to be biocompatible andnon-toxic in a variety of physiological applications. The preferredconcentrations of the polymer are 5% to 35% w/w, more preferably 5% to20% w/w. The polymer can be dissolved in a variety of aqueous solutions,but water is preferred.

The preferred polymer can be generally expressed as compounds of theformula:PEG-(DCR-CG)_(n)

Where:

DCR is a degradation control region.

CG is a crosslinking group.

n 3

While the preferred polymer is a multi-armed structure, a linear polymerwith a functionality, or reactive groups per molecule, of at least threecan also be used. The utility of a given PEG polymer significantlyincreases when the functionality is increased to be greater than orequal to three. The observed incremental increase in functionalityoccurs when the functionality is increased from two to three, and againwhen the functionality is increased from three to four. Furtherincremental increases are minimal when the functionality exceeds aboutfour.

The uses of PEG polymers with functionality of greater than threeprovides a surprising advantage. When crosslinked with higherfunctionality PEG polymers, the concentration of albumin can be reducedto 25% and below. Past uses of difunctional PEG polymers requireconcentrations of albumin well above 25%, e.g. 35% to 45%. Use of lowerconcentrations of albumin result in superior tissue sealing propertieswith increased elasticity, a further desired result. Additionally, 25%human serum albumin, USP is commercially available from several sources,however higher concentrations of human serum albumin, USP are notcommercially available. By using commercially available materials, thedialysis and ultrafiltration of the albumin solution, as disclosed inthe prior art, is eliminated, significantly reducing the cost andcomplexity of the preparation of the albumin solution.

C. The Resulting Genus 10 Hydrogel Composition

Upon mixing the fundamental protein solution component with thefundamental polymer solution, the non-liquid, three-dimensional network(i.e., the hydrogel) is formed.

To minimize the liberation of heat during the crosslinking reaction, theconcentration of the crosslinking groups of the fundamental polymercomponent is preferably kept less than 5% of the total mass of thereactive solution, and more preferably about 1% or less. The lowconcentration of the crosslinking group is also beneficial so that theamount of the leaving group is also minimized. In a typical clinicalapplication, about 50 mg of a non-toxic leaving group is produced duringthe crosslinking reaction, a further desired result. In a preferredembodiment, the CG comprising an N-hydroxysuccinimide ester hasdemonstrated ability to participate in the crosslinking reaction withalbumin without eliciting adverse immune responses in humans.

The genus material composition is well tolerated by the body, withoutinvoking a severe foreign body response. Over a controlled period, thematerial is degraded by physiological mechanisms. Histological studieshave shown a foreign body response consistent with a biodegradablematerial, such as VICRYL™ sutures. As the material is degraded, thetissue returns to a quiescent state. The molecules of the degraded genushydrogel composition are cleared from the bloodstream by the kidneys andeliminated from the body in the urine. In a preferred embodiment of theinvention, the material loses its physical strength during the firstfifteen days, and totally resorbs in about four weeks.

III. Creating The Species Compositions

Species compositions are created from the genus composition bycontrolling the rate of gelation, or controlling the rate ofdegradation, or controlling the mechanical properties, or combinationsthereof. The controlled properties of the species permit the use of thegenus 10 material composition in diverse therapeutic indications.

A. Controlling the Rate of Gelation

The rate of gelation is optimally controlled by the selection of thecrosslinking group (CG) and the reaction pH. The concentration of the CGin the polymer solution, and the concentration of nucleophilic groups inthe protein solution also can be used to control the rate of gelation,however changes in these concentrations typically result in changes inthe mechanical properties of the hydrogel, as well as the rate ofgelation.

The electrophilic CG is responsible for the crosslinking of the albumin,as well as binding the hydrogel to the surrounding tissue. The CG can beselected to selectively react with thiols, selectively react withamines, or react with thiols and amines. CG's that are selective tothiols include vinyl sulfone, N-ethyl maleimide, iodoacetamide, andorthopyridyl disulfide. CG's that are selective to amines includealdehydes. Non-selective electrophilic groups include active esters,epoxides, oxycarbonylimidazole, nitrophenyl carbonates, tresylate,mesylate, tosylate, and isocyanate. The preferred CG's are activeesters, more preferred, an ester of N-hydroxysuccinimide. The activeesters are preferred since they react rapidly with nucleophilic groupsand have a non-toxic leaving group.

The rate of gelation can also be controlled by the selection of thereaction pH. At a lower pH, a larger fraction of nucleophilic groups isunavailable for reaction with the electrophile. At higher pH's, a largerfraction of nucleophilic groups is available for reaction with theelectrophile. Ultimately, pH controls the concentration of nucleophilicgroups that are available for reaction. The reaction pH is a usefulmechanism to control the rate of gelation as it controls the ratewithout affecting the mechanical properties of the resulting hydrogel.

The reaction pH is optimally controlled by the buffer composition andconcentration. Preferred buffer systems are sodium phosphate and sodiumcarbonate, which can alone or in combination provide high or low pHbuffers. A high pH buffer is preferred when high rates of gelation aredesired. A low pH buffer is preferred for slower rates of gelation. Thebuffer concentration also plays a significant role in the rate ofgelation. Because of the crosslinking, a leaving group is formed. In thepreferred embodiment, the leaving group is acidic. So as the reactionproceeds, the pH drops and the rate of gelation slows. At higher bufferconcentrations, the fast rate of gelation is sustained over the lengthof the reaction. At lower concentrations, the buffer system is saturatedand no longer functions.

Through the selection of the CG and the reaction pH, the transformationof the material from a liquid to a solid can be controlled from lessthan 1 second to greater than 10 hours, more preferably less than 1second to 10 minutes, and most preferably less than 1 second to 2minutes.

It may be desirable to monitor the progression of the cross-linking. Forexample, when using Species 1 (to seal vascular puncture sites), it isdesirable to know when the composition is semi-solid, which indicatesthat it is time to remove the catheter/introducer and apply pressure tothe puncture site. It is also desirable to next know when thecomposition is solid, which indicates that pressure to the puncture sitecan be removed. The transitions from liquid to semi-solid and then fromsemi-solid to solid can be determined by timing the reaction.

The pH of the composition (protein and polymer) changes as cross-linkingprogresses (see FIG. 6). The change in pH during cross-linking for agiven composition can be empirically determined using aspectophotometer. The pH is high (e.g., pH 9 to 10) when the polymer isliquid (time t1 in FIG. 6). The pH is lower (e.g., pH 7) (time t3 inFIG. 6) when the composition is solid. The pH is at an intermediatevalue (e.g., pH 8) when the composition is in transition between liquidand solid (time t2 in FIG. 6). The composition can include one or morecolorimetric pH indicators to indicate, by color changes, theprogression of the gelation.

For example, xylenol blue exhibits a purple color at pH 9–10, a yellowcolor at pH 8, and a yellow color at pH 7. Phenol red exhibits a redcolor at pH 9–10, a red color at pH 8, and a yellow color at pH 7. Byincluding a mixture of xylenol blue and phenol red in the composition,the composition, as it cross-links, will exhibit a purple/blue color(mixture of purple and red) at time t1 (pH greater than 9), indicating aliquid state; an orange color (mixture of yellow and red) at time t2 (pHabout 8), indicating a semi-solid state; and a yellow color (mixture ofyellow and yellow) at time t3 (pH about 8), indicating a solid state.

As another example (in which lower pH values can be differentiated),phenolphthalein or o-cresolphthalein exhibit a red color at pH 9–10 andexhibit are clear color (i.e., are “colorless”) at pH 8 and below.Bromothymol blue exhibits a blue color at pH 7 and above, and a yellowcolor at pH 6 and below. By including a mixture of phenolphthalein (oro-cresolphthalein) and bromothymol blue, the composition, as itcross-links, will exhibit a purple/reddish color (mixture of purple andred) when the pH is greater than 9, indicating a liquid state; a bluecolor (mixture of clear and blue) when the pH is about 8), indicating asemi-solid state; and a yellow color (mixture of clear and yellow) whenthe pH is about 6–7, indicating a solid state.

B. Controlling the Rate of Degradation

The rate of degradation is controlled by the degradation control region(DCR), the concentration of the CG's in the polymer solution, and theconcentration of the nucleophilic groups in the protein solution.Changes in these concentrations also typically result in changes in themechanical properties of the hydrogel, as well as the rate ofdegradation.

The rate of degradation is best controlled by the selection of thechemical moiety in the degradation control region, DCR. If degradationis not desired, a DCR can be selected to prevent biodegradation or thematerial can be created without a DCR. However, if degradation isdesired, a hydrolytically or enzymatically degradable DCR can beselected. Examples of hydrolytically degradable moieties includesaturated di-acids, unsaturated di-acids, poly(glycolic acid),poly(DL-lactic acid), poly(L-lactic acid),poly(□-caprolactone),poly(.-valerolactone), poly(.-butyrolactone), poly(amino acids),poly(anhydrides), poly(orthoesters), poly(orthocarbonates), andpoly(phosphoesters). Examples of enzymatically degradable DCR's includeSEQ ID NO 1 Leu-Gly-Pro-Ala (collagenase sensitive linkage) andGly-Pro-Lys (plasmin sensitive linkage). It should also be appreciatedthat the DCR could contain combinations of degradable groups, e.g.poly(glycolic acid) and di-acid.

Through the selection of the DCR, the transformation of the materialfrom a solid hydrogel to a degraded liquid can be controlled from aslittle as 1 day to greater than 500 days, more preferably 5 days to 30days.

C. Controlling the Mechanical Properties

Desired mechanical properties of the hydrogel include cohesive strength,adhesive strength, and elasticity. Through the selection of thefunctionality, concentration, and molecular weight of the protein andpolymer, the mechanical properties of the hydrogel can be adjusted tosuit a variety of clinical needs.

The mechanical properties of the hydrogel are controlled, in part, bythe number of crosslinks in the hydrogel network as well as the distancebetween crosslinks. Both the number of crosslinks and the distancebetween crosslinks are dependent on the functionality, concentration,and molecular weight of the polymer and the protein.

Functionality, or the number of reactive groups per molecule, affectsthe mechanical properties of the resulting hydrogel by influencing boththe number of and distance between crosslinks. As discussed previously,the utility of a given polymer significantly increases when thefunctionality is increased to be greater than or equal to three. Theobserved incremental increase in functionality occurs when thefunctionality is increased from two to three, and again when thefunctionality is increased from three to four. By increasing thefunctionality of the polymer or protein at a constant concentration, theconcentration of crosslinking groups available for reaction areincreased and more crosslinks are formed. However, increased mechanicalproperties cannot be controlled with functionality alone. Ultimately,the steric hindrances of the protein or polymer to which the reactivegroups are attached predominate and further changes in the mechanicalproperties of the hydrogel are not observed. The effect of functionalityis saturated when the functionality reaches about four.

The concentration of the protein and polymer also affect the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the protein and polymerconcentration increases the number of available crosslinking groups,thereby increasing the strength of the hydrogel. However, decreases inthe elasticity of the hydrogel are observed as the concentration of theprotein and polymer is increased. The effects on the mechanicalproperties by concentration are limited by the solubility of the proteinand polymer.

The polymer and protein molecular weight affects the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the molecular weight of theprotein and polymer decreases the number of available crosslinkinggroups, thereby decreasing the strength of the hydrogel. However,increases in the elasticity of the hydrogel are observed with increasingmolecular weight of the protein and polymer. Low molecular weightproteins and polymers result in hydrogels that are strong, but brittle.Higher molecular weight proteins and polymers result in weaker, but moreelastic gels. The effects on the mechanical properties by molecularweight are limited by the solubility of the protein and polymer.However, consideration to the ability of the body to eliminate thepolymer should be made, as large molecular weight polymers are difficultto clear.

IV. Exemplary Species Compositions

It should be appreciated that by adjusting the rate of gelation, themechanical properties of the resulting hydrogel, and the rate ofdegradation, the genus material composition can be adapted for use in avariety of medical indications.

The following species compositions and their therapeutic indications areincluded by way of example.

A. Species 1: Sealing of Vascular Puncture Sites

For sealing vascular puncture sites, a biomaterial formulation with agelation time of fifteen to sixty seconds is preferred, more preferablyfifteen to thirty seconds. This period allows the biomaterial to bedelivered through the delivery device and flow into the surfaceirregularities before solidification. The period before solidificationalso enhances patient safety when compared to alternatives in the priorart.

Collagen plugs or slurries have been previously used to seal vascularpuncture sites. However, if the collagen plug or slurry enters thevasculature, emboli downstream from the arteriotomy is a distinctpossibility. In contrast, preclinical studies have demonstrated thatemboli do not form if the a biomaterial of the Species 1 enters thebloodstream before solidification. Biomaterial of the Species 1 isdiluted in flowing blood to a point where emboli cannot be formed.Furthermore, the rate of gelation is reduced at the pH of flowing blood,further enhancing the dilution effect.

For this indication, the hydrogel Species 1 possesses sufficientadhesive strength to prevent dislodging from the arteriotomy. Thehydrogel Species 1 also has sufficient cohesive strength to preventrupture under arterial pressure, i.e., up to about 200 mm Hg. Thehydrogel Species 1 also seals the arteriotomy for up to 15 dayspost-application before loss of mechanical properties throughdegradation, and completely degrades by 30 days post-application.

The following is a representative composition for Species 1:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylglutarate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with300 mM dibasic sodium phosphate, USP.

B. Species 2: Sealing Tissue from Blood Leaks

Preclinical studies have demonstrated that a biomaterial of Species 2 iseffective in controlling diffuse organ bleeding. The bleeding is notcontrolled via physiological interactions with the clotting cascade, butrather the biomaterial of Species 2 mechanically seals the tissue tocontrol the bleeding.

For this indication, the biomaterial formulation of the Species 2possesses an instantaneous gelation time. To achieve hemostasis in thisindication, the biomaterial Species 2 solidifies before being removedfrom the site by gravity and/or diluted by flowing blood. The resultinghydrogel Species 2 also possesses sufficient adhesive strength toprevent dislodging from the wound and sufficient cohesive strength toprevent rupture under arterial pressure, up to about 200 mm Hg. Thehydrogel Species 2 seals the wound for up to 15 days post-applicationbefore loss of mechanical properties through degradation, and completelydegrades by 30 days post-application.

The following is a representative composition for Species 2:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylsuccinate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with250 mM sodium carbonate and 50 mM sodium bicarbonate.

C. Species 3: Sealing Tissue from Gas Leaks

Preclinical studies have demonstrated that Species 3 is effective incontrolling air leaks from the lung. The biomaterial of Species 3 formsa mechanical barrier over the suture or staple line.

In this indication, the biomaterial of Species 3 possesses aninstantaneous gelation time. To achieve sealing in this indication, thebiomaterial solidifies before being removed from the site by gravity.The resulting hydrogel Species 3 possesses sufficient adhesive strengthto prevent dislodging from the wound and sufficient cohesive strength toprevent rupture under physiological lung pressure. Hydrogel Species 3also exhibits sufficient elasticity to withstand repeated lunginflation. The hydrogel Species 3 seals the wound for up to 15 dayspost-application before loss of mechanical properties throughdegradation, and completely degrades by 30 days post-application.

The following is a representative composition for Species 3:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylsuccinate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with250 mM sodium carbonate and 50 mM sodium bicarbonate.

D. Species 4: Sealing Tissue from Liquid Leaks

To seal a liquid leak, the biomaterial of Species 4 forms a mechanicalbarrier over the wound, suture, or staple line. The biomaterial ofSpecies 4 possesses an instantaneous gelation time, to solidify beforebeing removed from the site by gravity. The resulting hydrogel Species 4exhibits sufficient adhesive strength to prevent dislodging from thewound and sufficient cohesive strength to prevent rupture underphysiological pressure. The hydrogel Species 4 seals for up to 15 dayspost-application before loss of mechanical properties throughdegradation and completely degrades by 30 days post-application.

The following is a representative composition for Species 4:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylsuccinate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with250 mM sodium carbonate and 50 mM sodium bicarbonate.

E. Species 5: Sealing Tissue from Solid Leaks

To seal a solid leak, the biomaterial of Species 5 forms a mechanicalbarrier over the wound, suture, or staple line. The biomaterial ofSpecies 5 has an instantaneous gelation time, to solidify before beingremoved from the site by gravity.

The resulting hydrogel Species 5 has sufficient adhesive strength toprevent dislodging from the wound and sufficient cohesive strength toprevent rupture under physiological pressure. The hydrogel Species 5seals the wound for up to 15 days post-application before loss ofmechanical properties through degradation, and is completely degraded by30 days post-application.

The following is a representative composition for Species 5:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylsuccinate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with250 mM sodium carbonate and 50 mM sodium bicarbonate.

F. Species 6: Prevention of Post-Operative Adhesions

The biomaterial of Species 6 coats the injured tissue surface,preventing the deposition of fibrin, and allows the formation of a newlayer of epithelial cells. For the prevention of post-operativeadhesions, the biomaterial of Species 6 is capable of beinglaparoscopically delivered, and possesses an instantaneous gelationtime. For this indication, the biomaterial Species 6 solidifies beforebeing removed from the site by gravity.

The resulting hydrogel Species 6 has sufficient adhesive strength toprevent dislodging from the wound. The hydrogel Species 6 adheres to thetissue for three to fifteen days post-application, preferably seven toten days, before significant amounts of degradation occur. Thebiomaterial of Species 6 completely degrades in five to 180 dayspost-application, preferably five to thirty days.

The following is a representative composition for Species 6:

Polymer component: 9% w/w 4-arm poly(ethylene glycol)tetra-succinimidylsuccinate, MW 10,000 in water for injection.

Protein component: 13% w/w human serum albumin, USP supplemented with250 mM sodium carbonate and 50 mM sodium bicarbonate.

G. Species 7: Repair of Tissue Voids

The biomaterial of Species 7 fills the tissue void and solidifies. Forrepair of tissue voids, a biomaterial of Species 7 possesses a gelationtime of approximately 5 seconds. The five second gelation time allowsthe formulation to enter the void, fill surface irregularities, achievehemostasis, and prevent the formation of air pockets inside thehydrogel. The resulting hydrogel Species 7 has sufficient adhesivestrength to prevent dislodging from the void and sufficient cohesivestrength to prevent rupture under venous pressure, up to 100 mm Hg. Thehydrogel Species 7 seals the wound for up to 15 days post-applicationbefore loss of mechanical properties through degradation and completelydegraded by 30 to 60 days post-application.

The following is a representative composition for Species 7:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylglutarate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with100 mM sodium carbonate and 50 mM sodium bicarbonate.

H. Species 8: Augmentation of Tissue

The biomaterial of Species 8 enhances the desired tissue and solidifies.For tissue augmentation, the biomaterial of Species 8 has a gelationtime of approximately 120 seconds, to allow the formulation to enter thesurface irregularities, to prevent the formation of air pockets insidethe hydrogel, and to allow the user to add or subtract volume to achievethe desired effect.

The resulting hydrogel Species 8 has sufficient adhesive strength toprevent dislodging from the tissue site. The hydrogel Species 8 does notdegrade for up to one year post-application.

The following is a representative composition for Species 8:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-propionicacid succinimidyl ester, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP

I. Species 9: Embolization of Arterio-Venous Malformations (AVM's)

The biomaterial of Species 9 is delivered as a liquid, but quickly setsup to a solid, embolizing the AVM. For embolization of AVM's, thebiomaterial of Species 9 has a gelation time of approximately 30 to 120seconds. The time before solidification allows the biomaterial ofSpecies 9 to fill the tortuous mass of blood vessels completely.

The resulting hydrogel Species 9 has sufficient adhesive strength toprevent dislodging from the tissue site. The degradation of the hydrogelSpecies 9 is not relevant, as the AVM is removed immediately afterembolization.

The following is a representative composition for Species 9:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylglutarate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin.

J. Species 10: Filling of Aneurysms

The biomaterial of Species 10 is delivered as a liquid, but quickly setsup to a solid to fill the aneurysm. For aneurysm filling, thebiomaterial of Species 10 exhibits a gelation time of approximately 5 to30 seconds. The time before solidification allows the formulation tofill the aneurysm completely.

The resulting hydrogel Species 10 has sufficient adhesive strength toprevent dislodging from the aneurysm and sufficient cohesive strength toprevent rupture under arterial pressure, up to about 200 mm Hg. Thehydrogel Species 10 does not degrade for up to one yearpost-application.

The following is a representative composition for Species 10:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-propionicacid succinimidyl ester, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin, USP supplemented with100 mM sodium carbonate and 50 mM sodium bicarbonate.

K. Species 11: Delivery of Pharmaceuticals

The biomaterial of Species 11 serves as the depot for the pharmaceuticalor vector. The resulting hydrogel Species 11 can be solidified directlyon the diseased tissue. For delivery of pharmaceuticals, a biomaterialof Species 11 has a gelation time of approximately 5 to 30 seconds. Theresulting hydrogel Species 11 has sufficient adhesive strength toprevent dislodging from the tissue and sufficient cohesive strength toprevent fragmentation. The degradation of the hydrogel Species 11 isdependent on the desired time frame for release of the pharmaceutical,ranging from 1 week to 1 year.

The following is a representative composition for Species 11:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylglutarate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin supplemented with 75 mMsodium carbonate and 75 mM sodium bicarbonate.

L. Species 12: Delivery of Cells

The biomaterial of Species 12 serves as the matrix for the cells to bedelivered. For delivery of cells, the biomaterial of Species 12 has agelation time of approximately 5 to 30 seconds.

The resulting hydrogel Species 12 has sufficient adhesive strength toprevent dislodging from the tissue and sufficient cohesive strength toprevent fragmentation. The degradation of the hydrogel Species 12 isdependent on the desired time frame for the cells to remodel the tissue,ranging from 1 week to 6 months.

The following is a representative composition for Species 12:

Polymer component: 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidylglutarate, MW 10,000 in water for injection.

Protein component: 25% w/w human serum albumin supplemented with 75 mMsodium carbonate and 75 mM sodium bicarbonate.

The following Table summarizes the gelation time, degradation time, andmechanical properties of the species 1 to 12 the composition genus 10.

TABLE 2 Principal Characteristics and Therapeutic Indications of Species1 to 12 of the Fundamental Genus Composition Spe- Gelation DegradationMechanical Therapeutic cies Time Time Properties Indication 1 15 to 6030 Days Adhesive Sealing of Seconds Strength: Vascular Prevent PunctureDislodgment Sites Cohesive Strength: Prevent Rupture Under ArterialPressure 2 Instantaneous 30 Days Adhesive Sealing Strength: Tissue fromPrevent Blood Leaks Dislodgment Cohesive Strength: Prevent Rupture UnderArterial Pressure 3 Instantaneous 30 days Adhesive Sealing Strength:Tissue from Prevent Gas Leaks Dislodgment Cohesive Strength: PreventRupture under Lung Pressure Elasticity: To Withstand Repeated LungInflation 4 Instantaneous 30 days Adhesive Sealing Strength: Tissue fromPrevent Liquid Dislodgment Leaks Cohesive Strength: Prevent Ruptureunder Physiologic Pressure 5 Instantaneous 30 days Adhesive SealingStrength: Tissue from Prevent Solid Leaks Dislodgment Cohesive Strength:Prevent Rupture under Physiologic Pressure 6 Instantaneous 5 to 30Adhesive Prevention Days Strength: of Post Prevent Operative DislodgmentAdhesions 7 5 30 to 60 Adhesive Repair of Seconds Days Strength: TissuePrevent Voids Dislodgment Cohesive Strength: Prevent Rupture underVenous Pressure 8 120 1 Year Adhesive Augmentation seconds Strength: ofPrevent Tissue Dislodgment 9 30 to N/A Adhesive Embolization 120Strength: of seconds Prevent AVM's Dislodgment 10 5 to 30 1 YearAdhesive Filling of Seconds Strength: Aneurysms Prevent DislodgmentCohesive Strength: Prevent Rupture Under Arterial Pressure 11 5 to 30 1Year Adhesive Delivery of Seconds Strength: Pharmaceuticals PreventDislodgment Cohesive Strength: Prevent Fragmentation 12 5 to 30 1 weekto 6 Adhesive Delivery of seconds months Strength: Cells PreventDislodgment Cohesive Strength: Prevent FragmentationVI. General Methodology for Species Development

FIG. 5 shows a flowchart illustrating a methodology 200 for developingspecies compositions based upon the composition genus 10.

The first step 202 is to select a desired clinical indication. Basedupon the therapeutic requirements of the selected clinical indication,steps 204, 206, and 208 are followed to identify, respectively, themechanical properties, the rate of gelation, and the rate of degradationsuited for the indication.

Upon identifying the mechanical properties desired for the indication, astep 210 is carried out to selectively select the components of thecomposition genus 10, so as to create a species having the desiredmechanical properties. As discussed previously, the mechanicalproperties can be selected through the concentration of the protein andthe polymer. Elasticity can be obtained through lower concentrations ofthe protein and the polymer and increasing molecular weight of thepolymer. Cohesive strength can be obtained through higher concentrationsof the protein and the polymer and decreasing molecular weight of thepolymer. Increased adhesive strength can be obtained by increasing theratio of the concentration of polymer to the concentration of theprotein. Until the buffer system is fully optimized, the evaluation ofthe mechanical properties at this step 210 should be performed after asuitable cross-linking period, to allow completion of the crosslinkingreaction.

Once the desired mechanical properties have been achieved, a step 212can be carried out to further selectively tailor the components of thecomposition genus 10 to create for the species the desired rate ofgelation. As discussed previously, the rate of gelation can be selectedwith the buffer system and the cross-linking group of the polymer.Increased rates of gelation can be achieved by using carbonate buffers,higher pH's, and higher buffer concentrations. Decreased rates ofgelation can be achieved by using phosphate buffers, lower pH's, andlower buffer concentrations. Once the desired rate of gelation has beenobtained, it should be verified that the desired mechanical propertiesare still present during the clinically relevant period.

Once the desired mechanical properties and rate of gelation have beenachieved, a step 214 can be carried out to further selectively tailorthe components of the composition genus 10 to create for the species thedesired rate of degradation. As discussed previously, the rate ofdegradation can be selected by changing the degradation control regionon the polymer portion of the genus composition. Increased rates ofdegradation can be achieved by using glycolide or lactide, whiledecreased rates of degradation can be achieved by using glutaric acid asthe degradation control region. A formulation that does not degrade canalso be achieved by elimination of the degradation control region. Oncethe desired rate of degradation has been obtained, it should be verifiedthat the desired mechanical properties and rate of gelation are stillmaintained.

A step 216 can now be conducted to evaluate the species in in vitromodels, if available. These models are used to verify the mechanicalproperties and rate of gelation in a clinically relevant manner. If theresults indicate that these properties need to be adjusted, they can berefined.

A final step 218 comprises in vivo experimentation. In the in vivoexperimentation, the biocompatibility, effectiveness, and rate ofdegradation of the species are confirmed.

VII. Exemplary Delivery Systems

The delivery systems 14 serve to mix the fundamental protein and polymersolution components intimately, using atomization, static mixers, orin-line channel mixing. The mixing technique employed depends upon therequirements of the particular therapeutic indication, and, inparticular, the gelation time and morphology of the treatment site.

A typical delivery system 14 for the genus material composition (seeFIG. 3) includes, in the first kit 16, first and second dispensingsyringes 60 and 62, in which the formative components of the genusmaterial composition are contained.

The first dispensing syringe 60 contains a concentration of bufferedprotein solution component 100. The protein solution is supplementedwith the appropriate buffers, sterile filtered, aseptically filled intothe syringe 60, and the syringe 60 is capped for storage prior to use.

The second dispensing syringe 62 contains an inert, electrophilic, watersoluble polymer component 102. The polymer component 102 is initiallypackaged prior to use in the second dispensing syringe 62 in an inertatmosphere (e.g., argon) in a stable, powder form.

In this arrangement, the first kit 16 includes a third syringe 104,which contains sterile water 106 for dissolution of the powder polymer102 just before mixing with the albumin component 100. In facilitatingmixing, a stopcock valve 108 is secured to the luer fitting 88 at thedispensing end of the second dispensing syringe 62. The dispensing end110 of the water syringe 104 couples to the stopcock valve 108, so thatthe water 106 can be mixed with the polymer 102 in the dispensingsyringe 62 prior to use.

As FIG. 3 also shows, the second kit 18 carries the materialintroducer/mixer 22. As FIG. 4 shows, the two dispensing syringes 60 and62 are snap-mounted on the material introducer/mixer 22. The materialintroducer/mixer 22 includes a joiner 84. The joiner 84 includes side byside female luer fittings 86. The female luer fittings 86 each receivesthe threaded male luer fitting 88 at the dispensing end of thedispensing syringes 60 and 62.

The joiner 84 includes interior channels 90 coupled to the female luerfittings 86. The channels 90 merge at a Y-junction into a single outletport 92. The joiner 84 maintains two fluids dispensed by the syringes 60and 62 separately until they leave the joiner 84. This design minimizesplugging of the joiner 84 due to a mixing reaction between the twofluids. A syringe clip 68 can be provided to ensure even application ofindividual solutions through the joiner 84.

The parts of the introducer/mixer 22 and joiner are made, e.g., bymolding medical grade plastic materials, such as polycarbonate andacrylic.

For those therapeutic indications where the species composition needs toundergo instantaneous gelation, or gelation within a matter of a fewseconds, and where the application site is exposed (e.g., Species 2, 3,4, 5, 6, 7, 8, 9), the material introducer/mixer 22 can include a mixingspray head 94 coupled to the joiner (see FIG. 4). Preferably, the kitcontains several interchangeable mixing spray heads 94, in case onemixing spray head 94 becomes clogged during use.

The mixing spray head 94 may be variously constructed and comprise aconventional spray head.

Alternatively, the material introducer/mixer 22 can include a cannula152, which, in use, can be coupled to the joiner.

For those therapeutic indications where the species composition needs toundergo a longer period of gelation, and where access is required to asubsurface tissue site (e.g., Species 1 and 10), the materialintroducer/mixer 22 can include a catheter tube assembly 26 (see FIG. 3)that couples to the joiner 84. The catheter tube assembly 24 includes,at its distal end, a circumferentially spaced array of nozzles 34. Thebarrier material is conveyed in liquid form and dispensed in acircumferential manner through the nozzles 34 at the puncture site.

Expressed in tandem from the dispensing syringes 60 and 62, which aremechanically linked together by the joiner 84, the two fundamentalcomponents of the genus material composition come into contact in theliquid state either in the mixing spray head 94, or the cannula 152, orin the catheter tube assembly 26. Atomization of the two componentsoccurs as they are dispersed through the mixing spray head 94. Passageof the liquid components through the cannula 152 or catheter tube willchannel-mix the materials. Either by atomization or channel mixing, theliquid components are sufficiently mixed to immediately initiate thecross-linking reaction.

The material introducer/mixer 22 allows the physician to uniformlydispense the two components in a liquid state from the dispensingsyringes 60 and 62. The material introducer/mixer 22 also mixes thecomponents while flowing in the liquid state from the dispensingsyringes 60 and 62.

Further details of delivery systems for those species applied byspraying on exposed tissue sites are found in copending U.S. patentapplication Ser. No. 09/283,535, filed Apr. 1, 1999, and entitled“Compositions, Systems, And Methods For Arresting or ControllingBleeding or Fluid Leakage in Body Tissue,” which is incorporated hereinby reference.

Further details of delivery systems for those species introduced bycatheter-based systems are found in copending U.S. patent applicationSer. No. 09/188,083, filed Nov. 6, 1998 and entitled “Compositions,Systems, and Methods for Creating in Situ, Chemically Cross-linked,Mechanical Barriers,” which is likewise incorporated herein byreference. For example, when used to deliver the Species 1 materialcomposition, a 5.5 Fr catheter tube can be used to seal arteriotomiesfrom 5 Fr to 10 Fr, without filling the tissue track with the Species 1material composition. The Species 1 material composition is deliveredadjacent to the arteriotomy, while the delivery device prevents theliquid from filling the tissue track. This feature ensures that theSpecies 1 material composition remains at the arteriotomy for maximumefficacy.

The features of the invention are set forth in the following claims.

1. A method comprising providing a protein solution, providing a polymersolution including a derivative of a hydrophilic polymer with afunctionality of at least three, mixing the protein solution and thepolymer solution, the protein solution and the polymer solutioncross-linking to form a non-liquid hydrogel composition having adegradation time in situ that is from 1 week to 6 months, selecting atherapeutic agent comprising cells, and causing the therapeutic agent tobecome incorporated into the hydrogel composition, the degradation timebeing within a desired time frame for the cells to remodel tissue.
 2. Amethod comprising providing a protein solution, providing a polymersolution including a derivative of a hydrophilic polymer with afunctionality of at least three, the polymer solution comprising 17% w/w4-arm poly(ethylene glycol) tetra-succinimidyl glutarate, MW 10,000, inwater, mixing the protein solution and the polymer solution, the proteinsolution and the polymer solution cross-linking to form a non-liquidhydrogel composition, selecting a therapeutic agent, and causing thetherapeutic agent to become incorporated into the hydrogel composition.3. A method comprising providing a protein solution comprising 25% w/walbumin supplemented with 75 mM sodium carbonate and 75 mM sodiumbicarbonate, providing a polymer solution including a derivative of ahydrophilic polymer with a functionality of at least three, mixing theprotein solution and the polymer solution, the protein solution and thepolymer solution cross-linking to form a non-liquid hydrogelcomposition, selecting a therapeutic agent, and causing the therapeuticagent to become incorporated into the hydrogel composition.
 4. A methodas in claim 2 or 3 wherein the therapeutic agent is a pharmaceutical. 5.A method as in claim 4 wherein the hydrogel composition has adegradation time in situ that is within a desired time frame for releaseof the selected pharmaceutical.
 6. A method as in claim 5 wherein thedesired time frame is from 1 week to 1 year.
 7. A method as in claim 1wherein the polymer solution comprises 17% w/w 4-arm poly(ethyleneglycol) tetra-succinimidyl glutarate, MW 10,000, in water.
 8. A methodas in claim 1 wherein the protein solution comprises 25% w/w albuminsupplemented with 75 mM sodium carbonate and 75 mM sodium bicarbonate.9. A method as in claim 8 wherein the albumin is human serum albumin.10. A method as in claim 1 or 2 or 3 wherein the protein is a naturalprotein.
 11. A method as in claim 1 or 2 or 3 wherein the protein is arecombinant protein.
 12. A method as in claim 2 or 3 wherein thetherapeutic agent is cells.
 13. A method as in claim 12 wherein thehydrogel composition has a degradation time in situ that is within adesired time frame for the cells to remodel tissue.
 14. A method as inclaim 13 wherein the desired time frame is from 1 week to 6 months. 15.A method as in claim 1 or 2 or 3 wherein the hydrogel composition has agelation time in situ of 5–30 seconds.
 16. A method as in claim 1 or 2or 3 wherein the hydrogel composition comprises a three-dimensionalnetwork.
 17. A method as in claim 16 wherein the network degrades overtime back to a liquid form.
 18. A method as in claim 3 wherein thealbumin is human serum albumin.